Early Stages of Golgi Vesicle and Tubule Formation Require Diacylglycerol

Abstract

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed. INTRODUCTIONFormation of buds to generate intracellular transport vesicles from membranes such as Golgi cisternae involves both coat binding and local lipid conversion (for reviews and theoretical models, see Kirchhausen, 2000;Shemesh et al., 2003;Weiss and Nilsson, 2003;Bethune et al., 2006). For COPI vesicles, formation of buds is initiated by the small GTPase ARF1 (ADP-ribosylation factor 1) which, in its GTPconferred conformation, drives coatomer recruitment from the cytosol to both Golgi and pre-Golgi membranes (Palmer et al., 1993). Indeed, ARF1 and coatomer are sufficient for both bud and vesicle formation as evidenced in in vitro experiments using liposomes forming coated vesicles in a controlled manner (Spang et al., 1998). Addition of ARF-GAP1, a GTPase-activating protein for ARF1, then yielded uncoated vesicles of the expected size of ϳ50-60 nm in diameter (Reinhard et al., 2003).The situation in biological membranes is likely more refined involving additional as well as alternative components to promote or prevent vesicle formation such that Golgi function is maintained. Here, both ARF1 and ARFGAP1 have been implicated in vesicle formation through direct or indirect modulation of lipid synthesis such that bud formation and membrane fission are promoted. For example, ARF1 stimulates the production of phosphatidic acid (PA) from phosphatidylcholine (PC; Brown et al., 1993;Cockcroft et al., 1994) through the activation of phospholipase D (PLD) in a nucleotide (GTP)-specific manner Houle et al., 1995;Ktistakis et al., 1995). Such ARF1-mediated PLD stimulation results in an increased vesicle production (Ktistakis et al., 1996;Chen et al., 1997). This ability of ARF1 to stimulate lipid formation in the Golgi apparatus offers a possibility to mechanistically link lipid conversion with coat recruitment. PA may also be converted to diacylglycerol (DAG) and the ratio between DAG and PC seems to influence protein transport through the Golgi apparatus in yeast (Rivas et al., 1999). PA can also be synthesized from lysophosphatidic acid (LPA) by a LPA acyltransferase-dependent pathway through...

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

“…Digital images were obtained through a side-mounted MegaView III TEM CCD camera and quantified according to Ref. 40.…”

Phospholipid Synthesis Participates in the Regulation of Diacylglycerol Required for Membrane Trafficking at the Golgi Complex

“…5 is needed at the trans-Golgi network for the recruitment of protein kinase D and post-Golgi protein transport (7) and at the cis-Golgi for the biogenesis of coat protein complex I-coated vesicles (8,9). Several pharmacological agents were used to show this requirement of DAG and also to examine its source.…”

Characterization of the Yeast Actin Patch Protein App1p Phosphatidate Phosphatase

“…Differential effects of N-ethylmaleimide on Lpp1p (sensitive to inhibition) and Dpp1p (insensitive to inhibition) PAP activities might also be used to differentiate these Mg 2ϩ -independent enzymes in yeast extracts (36,41), yet the sensitivity to N-ethylmaleimide inhibition cannot be used to differentiate Mg 2ϩ -dependent and Mg 2ϩ -independent PAP activities in yeast. Likewise, inhibition by propranolol, a nonspecific ␤-blocker (80) that is commonly used to establish roles of PAP enzymes in various physiological processes (63,103,104), cannot be used to distinguish between App1p and Pah1p PAP enzymes or to distinguish between Mg 2ϩ -dependent and Mg 2ϩ -independent PAP enzymes because these PAP enzymes are sensitive to this reagent (41, 77,82,105).…”